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Sandermann, H., Jr., Tisue, G . T., and Grisebach, H. (1968), Biochim. Biophys. Acta 165,550. Schabel, F. M., Jr. (1968), Chemotherapy 13,321. Schardein, J. L., and Sidwell, R. W. (1968), Antimicrob. Ag. Chemotherap., 158. Seto, H., Otake, N., and Yonehara, H. (1968), Agr. Biol. Chem. (Tokyo)32,1299. Sidwell, R. W., Arnett, G . , and Schabel, F. M., Jr. (1969), Progr. Antimicrob. Anticancer Chemother., Proc. Int. Congr. Chemother., 6th Meeting, 2,44. Sidwell, R. W., Dixon, G. J., Schabel, F. M., Jr., and Kaump, D. H. (1968), Antimicrob. Ag. Chemorher., 148. Sloan, B. J., Miller, F. A., Ehrlich, J., McLean, I. W., Jr., and Machamer, H. E. (1968), Antimicrob. Ag. Chemother., 161. Suhadolnik, R. J. (1970), Nucleoside Antibiotics, New York,
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N. Y., John Wiley & Sons, Inc. Suhadolnik, R. J., and Chock, S. P. (1971), 162nd National Meeting of the American Chemical Society, Washington, D. C. Suhadolnik, R. J., Chock, S. P., and Uematsu, T. (1971), 162nd National Meeting of the American Chemical Society, Washington, D. C. Suhadolnik, R. J., and Farmer, P. B. (1971), 162nd National Meeting of the American Chemical Society, Washington, D. C. Suhadolnik, R. J., Weinbaum, G . ,and Meloche, H. P. (1964), J. Amer. Chem. SOC.86,948. Tono, H., and Cohen, S. S. (1962), J. Biol. Chem. 237,1271. Uematsu, T., and Suhadolnik, R. J. (1972), Biochemistry (in press). Volk, W. A. (1959),J . Biol. Chem. 234, 1931.
Phylogeny of Hemoglobins. p Chain of Frog (Rana esculenta) Hemoglobin* Jean-Pierre Chauvet and Roger Achert
ABSTRACT: The amino acid sequence of the p chain of frog hemoglobin (Rana esculenta) has been studied. The five fragments produced by tryptic cleavage of the trifluoroacetylated, carboxymethylated p chain were first isolated. After removal of the trifluoroacetyl groups and further cleavage with trypsin, 15 peptides were purified and sequenced. The order of the tryptic peptides in each’ fragment was determined through chymotryptic peptides and the alignment of the fragments was established by isolating arginine-containing peptides from a chymotryptic digest of the p chain. The frog p chain comprises 140 residues. When the 26 known mammalian p, 8,and y chains
A
mong the proteins which were chosen for a study on molecular evolution, the hemoglobin family is one of the most attractive. Because hemoglobin is particularly abundant and easy to purify from red cells, the protein seems amenable to structural investigations in almost all vertebrates. On the other hand, the “molecule” is generally built with polypeptide From the Laboratory of Biological Chemistry, University of Paris VI, 96, Boulevard Raspail, Paris (6e), France. Receioed June 21, 1971. This study was supported in part by a grant from the DBlQgation a la Recherche Scientifique et Technique (66-00-146). Maps of chymotryptic peptides from tryptic fragments, tables of tryptic peptides subjected to Edman degradation and to chymotryptic hydrolysis, and tables of amino acid compositions and of yields of chymotryptic from tryptic fragments and from p chain will appear following these pages in the microfilm edition of this volume of the journal. Singles copies may be obtained from the Business Operations Office, Books and Journals Division, American Chemical Society, 1155 Sixteenth St., N.W., Washington, D . C . 20036, by referring to code number BIO-11-916. Remit check or money order for $3.00 for photocopy or $2.00 for microfiche. t To whom inquiries should be directed.
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and frog p chain are compared, 54 positions out of 146 are invariant in all the proteins. Of 47 amino acids involved in the interactions of p chain with either a chains or heme group, 30 are invariant. Two long sequences (28-40 and 96-108) seem particularly stable. The comparison of p chains from eutherians, metatherians (kangaroo), and amphibians (frog), which have diverged approximately 80, 130, and 320 million years ago, respectively, reveals that the number of amino acid substitutions is dependent but not proportional to time. These results are confronted with current concepts of evolution.
chains of 140-150 residues in length and determination of the complete amino acid sequence can be performed under rather good conditions with the current techniques of protein chemistry. However, separation of a and p chains turned out to be more difficult for lower vertebrates than for mammals and probably for this reason our knowledge on hemoglobins of lower vertebrates is very limited since to the present time only the LY chains of the chicken (Matsuda et al., 1970) and of the carp (Hilse et al., 1966) have fully been sequenced. The choice of hemoglobin as an evolutionary tracer is not only determined by practical reasons. In contrast to most enzymes, this protein takes in account a whole physiological function, the oxygen transport, and hence is directly subjected to selective pressure; the so-called phenotypic character on which selection acts is apparently related to a few structural genes and not to many genes as in a polymolecular function (Simpson, 1964). In another point of view, the vertebrates, except fishes, used dissolved oxygen in the first part of their life and aerial oxygen in the second part and the switch might be associated with a molecular change. This change has actually
0
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been detected in mammals, birds, and amphibians and to what extent it can reflect a t the biochemical level the recapitulation law of Haeckel deserves special attention. On the phylogenetic scale, amphibians are located between aquatic and terrestrial vertebrates. Because of this position, amphibian species seem of particular interest for the search of transition molecules. The example of neurohypophysial hormones is rather suggestive. Isotocin is present in bony fishes but mestocin appears in amphibians and remains in reptiles and birds (Acher et al., 1970). The comparison of amphibian proteins to those of fishes, on one hand, and those of reptiles on the other, might reveal significant changes. Furthermore in the case of hemoglobin, it is known that during the metamorphosis of tadpoles, the tadpole protein is replaced by an adult protein (Moss and Ingram, 1968) and the comparison of the first to fish hemoglobins could be very instructive. The present work is devoted to Rana esculenta hemoglobin. R.esculenta is a current European frog which is used for food and the collection of material in large amounts is relatively easy. The purification of the major component of R.esculenta hemoglobin as well as the separation of cy and p chain has been described elsewhere (Chauvet and Acher, 1967b, 1968, 1971). The amino acid sequence of p chain has been determined and this paper gives a detailed description of the work, the results of which have been briefly reported (Chauvet and Acher, 1970). Material and Methods
p Chain and Derifiatiues. Hemoglobin from adult frog Rana esculenta was prepared as previously described by fractionated precipitation with ammonium sulfate and chromatography on carboxymethyl-Sephadex C-50. The chromatography resolved hemoglobin into two components which have apparently similar amino acid compositions but different electrophoretic mobilities (Chauvet and Acher, 1967b, 1971). The major component (hemoglobin 11) was used for this study. Reduced globin was subjected to countercurrent distribution and (Y and p chains were separated with a yield of 60-70z. From 100 ml of frog blood, about 1.0-1.5 g (60-100 pmole) of each chain can be obtained. The purity of the material was checked by starch gel electrophoresis and by N-terminal analysis (Chauvet and Acher, 1968, 1971). Carboxymethylation of the SH groups was performed according to Crestfield e f al. (1963). p chain (100 mg) is dissolved in 10 ml of 0.5 M Tris-HC1 buffer (pH 8.5) containing 8 M urea and 20 mg of EDTA. p-Mercaptoethanol (0.1 ml; Fluka) is added. After 4 hr, 268 mg of iodoacetic acid (BDH) dissolved in 1 ml of 1 N NaOH is added and the p H is maintained at 8.5 for 30 min by addition of 1 N sodium hydroxide with a Radiometer pH-Stat. Aminoethylation was carried out by adding 0.3 ml of ethylenimine (Fluka) in the place of iodoacetic acid, the pH being fixed a t 8.5 for 60 mn by addition of 1 N HCl according to Raftery and Cole (1963). The p H is lowered to 3.0 with 1 N HCI and carboxymethylated or aminoethylated @ chain is dialyzed for removing salts and urea, precipitated by acetone, redissolved in 10 ml of water, and lyophilized (yield for carboxymethylated p chain, 99 mg; for aminoethylated p chain, 96 mg). Trifluoroacetylation of carboxymethylated p chain was performed according to Goldberger and Anfinsen (1962). Carboxymethylated p chain (100 mg) is dissolved in 10 ml of water and the pH is brought to 9.95 with 2 N KOH. S-Ethyl trifluorothioacetate (0.5 ml) is added and the pH is maintained at 9.95 for 90 min with 2 N K O H by using a Radiometer pH-
Stat. The pH is then lowered to 2.5 with 12 N HC1 and the derivative precipitates. The product is recovered by centrifuging, washed three times with 20 ml of 0.01 N HCl, suspended in 10 ml of water, and lyophilized (yield 103 mg). For removing the trifluoroacetyl groups, the derivatives are treated with 1 M piperidine (pH 12.4) at 5 " for 2 hr (Goldberger and Anfinsen, 1962). Electrophoresis. Starch gel electrophoresis is used for checking the homogeneity of p chain. Gel is prepared with a formate buffer (pH 1.9) (Muller, 1960) and staining is carried out with Amido Schwarz 10B (Merck). Paper electrophoresis is employed for checking the purity of large peptide fragments; Whatrnann No. 3MM paper in a buffer pH 3.7 (pyridineacetic acid-water, 1: 10: 298, v/v) is used and staining is carried out with 0.1 % ninhydrin in alcohol. Enzymatic Cleamges. @ chain is split by trypsin or chymotrypsin in order to obtain fragments for amino acid sequence determination. Trypsin (twice crystallized Worthington, batch TRSF 6144-5) (EC 3.4.4.4) is used with a n enzyme: substrate weight ratio 1:100 for 3 hr in 0.1 M ammonium bicarbonate (pH 8.0) a t 37". Two equal fractions of the enzyme are added at times 0 and 90 min. Selective cleavages a t arginine residues are obtained by using trifluoroacetylated carboxymethylated p chain. Chymotrypsin (three-times crystallized Worthington, batch DCI 6078) (EC 3.4.4.5) is employed with an enzyme:substrate weight ratio 2 : l O O for 3 hr in 0.1 M ammonium bicarbonate (pH 8.0) a t 37". Purification of Peptide Fragments. For fractionating the mixture of the large fragments produced by trypsin hydrolysis of the trifluoroacetylated carboxymethylated @ chain, gel filtration on columns of either Sephadex G-50 (4 X 100 cm) or Sephadex G-25 (2 X 90 cm) is used with 0.1 M acetic acid. The flow (50 and 15 ml per hr) is fixed with a Milton Roy pump. Fractions of 5 ml are collected and peptides are detected by absorbance at 232 and 280 nm with a Beckman D U spectrophotometer. When two components are not resolved by gel filtration on Sephadex G-50, the separation is completed either by another gel filtration on Sephadex G-25 or by paper electrophoresis. Isolation of tryptic or chymotryptic peptides is carried out by using the fingerprinting technique (Katz et al., 1959) under the conditions described by Baglioni (1961). High-voltage electrophoresis at 35 V/cm on Whatman No. 3MM paper with a pyridine-acetate buffer (pH 6.4; pyridine-acetic acid-water 10:0.4:90, v/v) in cooled tanks and descending chromatography in a n isoamyl alcohol-pyridine-water solvent (30:30:35, v/v) are performed. Staining is carried out with ninhydrin (0.1 in alcohol) or with reagents specific for arginine (Acher and Crocker, 1952), tyrosine (Acher and Crocker, 1952), histidine (Sanger and Tuppy, 1951), and tryptophan (Smith, 1953). For preparative purposes, staining is performed with dilute ninhydrin (0.01% in alcohol); the spots are cut, washed with ether, and eluted with 0.1 M acetic acid. The materials are kept frozen a t -2OO and used either for amino acid analysis or structural studies. Amino Acid Analysis and Sequence Techniques. Aliquots of peptides (100-200 nmoles) are hydrolyzed in evacuated, sealed tubes with 6 N HC1 at 110" for 24, 48, or 72 hr. After removal of HCl by evaporation in a desiccator with sodium hydroxide, hydrolysates are analyzed according to Spackman et al. (1958) on a Spinco automatic analyzer Model 120B fitted with a high-sensitivity colorimeter. N-Terminal sequences are determined by using the dinitrofluorobenzene technique of Sanger (Chauvet et af., 1966) and the stepwise degradation procedure of Edman in the paper strip variant BIOCHEMISTRY,
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